Probes for INS

An early inflammatory insult is the most recognized risk factor associated with neurodevelopmental psychiatric disorders, even more so than genetic variants. Notably, complement component 4 (C4), a molecule involved in inflammatory responses, has been strongly associated with schizophrenia (SZ) and its role in other neurodevelopmental disorders, such as autism (ASD), is an area of active investigation. However, while C4 in SZ has been implicated in the context of synaptic pruning, little is known about its neuroinflammatory role. The subventricular zone (SVZ) is a region heavily involved in neurodevelopment and neuroimmune interactions through the lifespan; thus, it is a region wherein C4 may play a vital role in disease pathology. Using in situ hybridization with radioactive riboprobes and RNAscope, we identified robust astrocytic expression of C4 in the SVZ and in the septum pellucidum. C4 was also expressed in ependyma, neurons, and Ki67+ progenitor cells. Examination of mRNA levels showed elevated C4 in both ASD and SZ, with higher expression in SZ compared to controls. Targeted transcriptomic analysis of inflammatory pathways revealed a strong association of complement system genes with SZ, and to a lesser extent, ASD, as well as generalized immune dysregulation without a strong association with known infectious pathways. Analysis of differentially expressed genes (DEGs) showed that ASD DEGs were enriched in adaptive immune system functions such as Th cell differentiation, while SZ DEGs were enriched in innate immune system functions, including NF-κB and toll like receptor signaling. Moreover, the number of Ki67+ cells was significantly higher in ASD compared to SZ and controls. Taken together, these results support a role for C4 into inflammatory-neuroimmune dysregulation observed in SZ and ASD pathology.

Following injury in the central nervous system, a population of astrocytes occupy the lesion site, form glial bridges and facilitate axon regeneration. These astrocytes originate primarily from resident astrocytes or NG2+ oligodendrocyte progenitor cells. However, the extent to which these cell types give rise to the lesion-filling astrocytes, and whether the astrocytes derived from different cell types contribute similarly to optic nerve regeneration remain unclear. Here we examine the distribution of astrocytes and NG2+ cells in an optic nerve crush model. We show that optic nerve astrocytes partially fill the injury site over time after a crush injury. Viral mediated expression of a growth-promoting factor, ciliary neurotrophic factor (CNTF), in retinal ganglion cells (RGCs) promotes axon regeneration without altering the lesion size or the degree of lesion-filling GFAP+ cells. Strikingly, using inducible NG2CreER driver mice, we found that CNTF overexpression in RGCs increases the occupancy of NG2+ cell-derived astrocytes in the optic nerve lesion. An EdU pulse-chase experiment shows that the increase in NG2 cell-derived astrocytes is not due to an increase in cell proliferation. Lastly, we performed RNA-sequencing on the injured optic nerve and reveal that CNTF overexpression in RGCs results in significant changes in the expression of distinct genes, including those that encode chemokines, growth factor receptors, and immune cell modulators. Even though CNTF-induced axon regeneration has long been recognized, this is the first evidence of this procedure affecting glial cell fate at the optic nerve crush site. We discuss possible implication of these results for axon regeneration.

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to simultaneously assess transcription and chromatin accessibility in EGCs undergoing spontaneous neurogenesis in culture, as well as small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. A subset of in vivo EGCs, highly enriched within the myenteric ganglia and that persist into adulthood, have a gene expression program and chromatin state consistent with neurogenic potential. These results clarify the mechanisms underlying EGC potential for neuronal fate transition.

How the vascular and neural compartment cooperate to achieve such a complex and highly specialized structure as the central nervous system is still unclear. Here, we reveal a crosstalk between motor neurons (MNs) and endothelial cells (ECs), necessary for the coordinated development of MNs. By analyzing cell-to-cell interaction profiles of the mouse developing spinal cord, we uncovered semaphorin 3C (Sema3C) and PlexinD1 as a communication axis between MNs and ECs. Using cell-specific knockout mice and in vitro assays, we demonstrate that removal of Sema3C in MNs, or its receptor PlexinD1 in ECs, results in premature and aberrant vascularization of MN columns. Those vascular defects impair MN axon exit from the spinal cord. Impaired PlexinD1 signaling in ECs also causes MN maturation defects at later stages. This study highlights the importance of a timely and spatially controlled communication between MNs and ECs for proper spinal cord development.

Ataxia-telangiectasia (A-T) is a genetic disorder caused by the lack of functional ATM kinase. A-T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A-T remains elusive. Here, we utilize human pluripotent stem cell-derived cortical brain organoids to study A-T neuropathology. Mechanistically, we show that the cGAS-STING pathway is required for the recognition of micronuclei and induction of a senescence-associated secretory phenotype (SASP) in A-T olfactory neurosphere-derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self-DNA-triggered SASP expression in A-T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A-T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A-T and constitutes a novel therapeutic target for treating neuropathology in A-T patients.

There is a growing amount of data uncovering the cellular diversity of the pulmonary circulation and mechanisms governing vascular repair after injury. However, the molecular and cellular mechanisms contributing to the morphogenesis and growth of the pulmonary vasculature during embryonic development are less clear. Importantly, deficits in vascular development lead to significant pediatric lung diseases, indicating a need to uncover fetal programs promoting vascular growth. To address this, we used a transgenic mouse reporter for expression of Cxcl12, an arterial endothelial hallmark gene, and performed single-cell RNA sequencing on isolated Cxcl12-DsRed+ endothelium to assess cellular heterogeneity within pulmonary endothelium. Combining cell annotation with gene ontology and histological analysis allowed us to segregate the developing artery endothelium into functionally and spatially distinct subpopulations. Expression of Cxcl12 is highest in the distal arterial endothelial subpopulation, a compartment enriched in genes for vascular development. Accordingly, disruption of CXCL12 signaling led to, not only abnormal branching, but also distal vascular hypoplasia. These data provide evidence for arterial endothelial functional heterogeneity and reveal conserved signaling mechanisms essential for pulmonary vascular development.

The regenerative capacity of the mammalian heart is poor with one potential reason being that adult cardiomyocytes cannot proliferate at sufficient levels to replace lost tissue. During development and neonatal stages, cardiomyocytes can successfully divide under injury conditions; however, as these cells mature their ability to proliferate is lost. Therefore, understanding regulatory programs that can induce post-mitotic cardiomyocytes into a proliferative state is essential to enhance cardiac regeneration. Here we report the forkhead transcription factor, foxm1, is required for cardiomyocyte proliferation after injury through transcriptional regulation of cell cycle genes. Transcriptomic analysis of injured zebrafish hearts revealed that foxm1 expression is increased in border zone cardiomyocytes. Decreased cardiomyocyte proliferation and expression of cell cycle genes in foxm1 mutant hearts was observed, suggesting it is required for cell cycle checkpoints. Subsequent analysis of a candidate Foxm1 target gene, cenpf, revealed this microtubule and kinetochore binding protein is also required for cardiac regeneration. Moreover, cenpf mutants show increased cardiomyocyte binucleation. Thus, foxm1 and cenpf are required for cardiomyocytes to complete mitosis during zebrafish cardiac regeneration.